This application is a 371 of PCT/EP00/12977 filed Dec. 20, 2000.
This invention refers to new compounds which can chelate paramagnetic bi- and trivalent metal ions, their chelates with said metal ions and their use as contrast agents in magnetic resonance imaging (M.R.I.).
From a radiologist""s point of view, an improvement in the radiographic image, which means a better contrast enhancement between healthy and diseased tissues, is seen as an aid to the diagnosis which can be obtained through previous administration of suitable exogenous substances.
These substances cause a significant alteration of a specific characteristic, known as relaxivity, of the water protons belonging to the tissue under examination, when such protons are submitted to an external magnetic field.
These substances are known as contrast agents for M.R.I. A number of chelated complexes of linear and cyclic polyaminopolycarboxylic ligands with paramagnetic metals are known to be useful as M.R.I. contrast agents.
Said compounds generally derive from the two basic polyaminopolycarboxylic structures, namely diethylenetriaminopentaacetic acid (DTPA) and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA).
The compounds of the present invention are novel polyamino derivatives comprising at least one phosphonic residue as one of the binding site in the chelating agent structure.
Relaxivity (r1p) is an intrinsic property of paramagnetic complexes which characterizes their ability to increase the nuclear magnetic relaxation rate of vicinal protons. In the case of Gd(III) chelates with qxe2x89xa71, wherein q is the number of coordinated water molecules, a remarkable contribution to the increase in relaxation observed for water protons of the solvent derives from the exchange between the molecule(s) of bound water and the molecules of the remaining solvent (S. Aime et al., Chem. Soc. Rev., 1998, 27, 19).
This contribution (r1pis) is related to the relaxation time (T1M) and to the residence time (xcfx84M) of the protons of the water molecule(s) which are coordinated in the inner coordination sphere according to the following equation:       r          1      ⁢      p        is    =                    1.8        ·                  10                      -            5                              ⁢              xe2x80x83            ⁢      q              (                        T                      1            ⁢            M                          +                  τ          M                    )      
T1M receives contributions from the reorientation of the paramagnetic species, xcfx84R, through the residence time of the coordinated water protons, xcfx84M, and the electronic relaxation time of the metal ion, xcfx84S. Moreover, r1pis is the highest when T1M greater than xcfx84M (fast exchange conditions) and T1M is as short as possible.
A remarkable increase in r1p at the magnetic field values conventionally used in clinical practice has been up to now obtained, in different ways, mainly by decreasing molecular tumbling, with a consequent increase in xcfx84R. The expected increase in r1p has not, however, been observed due to the limiting effect caused by the residence time of water molecules, xcfx84M. A fine tuning of this parameter has become the primary object of current research in the M.R.I. field, as only xcfx84M values of about 30 ns would make it possible to completely exploit the decrease in T1M induced by the increase in xcfx84R. For this reason, the exchange rate values of water molecules in lanthanide (III) complexes are of paramount importance in the development of novel M.R.I. contrast agents. In fact, the residence time of the water molecule(s) coordinated to a Gd(III) complex plays a particularly important role, in that it directly contributes to the nucleus-electron dipolar interaction and controls the transfer efficiency of the paramagnetic effect to the water molecules of the solvent.
The above cited prior art contrast agents, generally comprising polyaminopolycarboxylic acid derivatives, have shown xcfx84M values generally comprised between 200 and 2500 ns, where such values are significantly higher than the 30 ns optimum one.
Optimization and harmonization of the above parameters are still remarkably important objects for everyone dealing with the development of novel M.R.I. contrast agents.
The present invention relates to polyamino derivatives comprising as the binding site in the structure of the chelating agent at least one phosphonic is residue, capable of causing an increase in the proton exchange rate and therefore advantageously low xcfx84M values.
More particularly, the object of the present invention is acyclic polyamino derivative chelating agents of formula (I), both in the racemic and the optically active forms, 
wherein
Y is a COOH group or a PO(OH)2 group, with the proviso that at least one Y group is xe2x95x90PO(OH)2;
R is a hydrogen atom, or xe2x80x94(CH2)mxe2x80x94Oxe2x80x94R2, (C1-C5)-alkyl-(C6-C10)-aryl or (C1-C5)-alkyl-heteroaryl whose aryl or heteroaryl moiety comprises 1 or 2 fused rings optionally substituted with one or more halogen atoms, OH groups, alkyl(C1-C5) groups and/or an OR3 group, wherein
R2 is (C1-C5)-alkyl-(C6-C10)-aryl, optionally substituted with one or more halogen atoms, OH and (C1-C5)-alkyl groups;
R3 is (C6-C10) aryl optionally substituted with one or more halogen atoms, OH and/or (C1-C5)-alkyl groups;
m ranges from 1 to 5;
R1 can have the same meanings as R with the proviso that when Y is PO(OH)2, R1 is selected from H, (CH2)mNH2, (CH2)mCOOH or an amido derivative thereof.
A further object of the invention are the chelates of said compounds of formula (I) with the bi- and trivalent ions of metal elements having atomic number ranging between 20 and 31, 39, 42, 43, 44, 49, or between 57 and 83, as well as the salts thereof with physiologically compatible organic bases selected from primary, secondary, tertiary amines or basic amino acids, or with inorganic bases whose cations selected from sodium, potassium, magnesium, calcium or mixtures thereof.
A further object of the present invention is the use of the compounds of formula (I), their complexes with paramagnetic metals and the physiologically compatible salts thereof for the preparation of pharmaceutical formulations for the imaging of organs and/or tissues of the human or animal body, by use of M.R.I.
Examples of (C1-C5)-alkyl-(C6-C10)-aryl groups comprise benzyl, phenethyl, naphthylmethyl wherein the aryl moiety is optionally substituted with one or more halogen atoms or OR3 groups wherein R3 is as defined above.
Examples of (C1-C5)-alkyl-heteroaryl groups comprise pyridylmethyl or indolylmethyl.
Examples of (C6-C10) aryl groups comprise phenyl or naphthyl optionally substituted with one or more halogen atoms, OH and/or (C1-C5)-alkyl groups.
Examples of (C1-C5) alkyl groups preferably comprise methyl, ethyl, isopropyl.
Preferred are compounds of formula (II), 
wherein 4 side carboxylic groups and a central phosphonic group are present and wherein
R and R have the above defined meanings.
Among compounds of formula (II), particularly preferred are those in which R1 is an hydrogen atom and R can assume all previously defined meanings.
Also preferred are the compounds of formula (III) 
wherein two side phosphonic groups and three carboxylic groups are present, and wherein
R1 has all the values defined above, as well as the compounds of general formula (IV), 
xe2x80x83wherein three phosphonic groups and two carboxylic groups are present and wherein
R has the values defined above.
Particularly preferred are the following compounds:
N,Nxe2x80x2-[(Phosphonomethylimino)di-2,1-ethanediyl]bis[N-carboxymethyl-L-phenylalanine];
[[4S-(4R*,12R*)]-4-Carboxy-5,11-bis(carboxymethyl)-1-phenyl-12-[(phenylmethoxy)methyl]-8-(phosphonomethyl)-2-oxa-5,8,11-triazatridecan-13-oic] acid;
N,Nxe2x80x2-[(Phosphonomethylimino)di-2,1-ethanediyl]bis[N-carboxymethyl-L-tryptophan];
N,N-Bis[2-[(carboxymethyl)(phosphonomethyl)amino]ethyl]-O-(4-hydroxyphenyl)-3,5-diiodo-L-tyrosine;
N,Nxe2x80x2-[(Phosphonomethylimino)di-2,1-ethanediyl]bis[N-(carboxy-methyl)-glycine];
N,Nxe2x80x2-[(Phosphonomethylimino)di-2,1-ethanediyl]bis[N-(phosphonome-thyl)glycine];
N,Nxe2x80x2-[[[3-Carboxy-1-phosphonopropyl]imino]di-2,1-ethanediyl]bis[N-(carboxymethyl)glycine];
4-Phenyl-N-[trans-4-[[[4-[bis[2-[bis(carboxymethyl)amino]ethyl]amino]-1-oxo-4-phosphonobutyl]amino]methyl]cyclohexylcarbonyl]-L-phenylalanina;
(3xcex2, 5xcex2, 7xcex1, 12xcex1)-3-[[4-[bis[2-[bis(carboxymethyl)amino]ethyl]amino]-1-oxo-4-phosphonobutyl]amino]-7,12-dihydroxycholan-24-oic acid;
N,Nxe2x80x2-[[[3-Amino-1-phosphonopropyl]imino]di-2,1-ethanediyl]bis[N-(carboxymethyl)glycine];
as well as the paramagnetic chelated complexes thereof and the physiologically compatible salts thereof.
Preferred chelates are those in which the bi- or trivalent metal ion is selected from Gd(3+), Dy(3+), Fe(3+), Fe(2+) and Mn(2+). Particularly preferred are Gd(3+) chelates.
Preferred cations of inorganic bases optionally suitable for salifying the chelated complexes of the invention particularly comprise the ions of alkali or alkaline-earth metals such as potassium, sodium, calcium, magnesium, and mixtures thereof.
Preferred cations of organic bases suitable for this purpose comprise, inter alia those obtained by protonation of primary, secondary and tertiary amines such as ethanolamine, diethanolamine, morpholine, glucamine, N-methylglucaimine, N,N-dimethylglucarmine.
Preferred cations of amino acids comprise, for example, those of lysine, arginine or ornithine.
The introduction of at least one phosphonic group as the binding site in the structure of the chelating agent unexpectedly provided contrast agents having an advantageous increase in the proton exchange rate and, therefore, particularly low xcfx84M values.
In particular, the chelated complexes of the invention are characterized by xcfx84M less than 100 ns values, preferably values between 10 and 100 ns, most preferably between 20 and 50 ns.
Among the various synthetic approaches to the compounds of the invention, the one preferred for the preparation of the compounds of formula (II), and particularly for the compounds in which R1 is xe2x95x90H and R has the meanings defined above in claim 1, is reported in the following Scheme 1: 
wherein R has the values defined above for compounds (I).
Briefly, the synthetic process of Scheme 1 comprises the following steps:
a) Esterification of a suitable amino acid. Said esterification can be advantageously carried out by reacting the amino acid with an alkyl acetate and an acid such as HClO4. In a variation of the process, the amino acid, previously N-protected by reaction with CBZCl , can be esterified by reaction with an alkyl halide, in the presence of a base such as K2CO3;
b) N-Alkylation of the resulting ester (intermediate 1) by reacting it with a suitable bromoacetate, such as tert-butyl bromoacetate. Said reaction is carried out in an organic solvent preferably selected among acetonitrile, THF, EtOAc and in the presence of a pH 8 buffer solution;
c) Bromoalkylation of the intermediate 2 by reacting it with trifluoromethanesulfonic acid 2-bromoethyl ester (intermediate 3) previously prepared from bromoethanol, trifluoromethanesulfonic anhydride and 2,6-lutidine. The bromoalkylation is carried out in an organic solvent suitably selected among, for example, toluene, acetonitrile, dichloroethane, and in the is presence of an amine selected among ethylenediamine, diisopropylethylamine, triethylamine, to give the intermediate 4. In a variation of the process of the invention, compound 4 can be alternatively prepared starting from the corresponding N-(2-hydroxyethyl) derivative, obtained as described in WO 98/05625, (incorporated herein by reference in its entirety), by reacting it with a brominating agent such as NBS, in the presence of triphenylphosphine;
d) Preparation of aminomethylphosphonic acid diethyl ester (intermediate 5) by direct condensation of tribenzylhexahydrotriazine with a suitable dialkyl phosphite and subsequent debenzylation by catalytic hydrogenation of the condensation product;
e) Bis alkylation of intermediate 5 by reaction with intermediate 4 and isolation of hexaester 6. In the process of the invention, the bis alkylation reaction is preferably carried out in an organic solvent such as acetonitrile, ethyl acetate, and in the presence of a pH 8 buffer solution;
f) Deprotection of the acidic functions of intermediate 6 and isolation of the acid chelating agent 7. Said deprotection can be obtained by reacting the hexaester with iodotrimethylsilane in an organic solvent, such as CH3CN.
A different synthetic approach for the preparation of the compounds of formula (II) in which, on the contrary, R is xe2x95x90H and R1 has the meanings defined above in claim 1, is reported in the following Scheme 1 bis: 
in which, as an example, is detailed the preparation of one of several preferred complex compounds of the invention.
The synthetic process of SCHEME 1bis essentially comprises the following steps:
a) Preparation of 2,2xe2x80x2-(Iminodi-2,1-ethanediyl)bis-1H-isoindole-1,3(2H)-dione) (intermediate 1) by reacting phthalic anhydride with diethylenetriamine in acetic acid;
b) N-alkylation of the bis-phthalimido derivative 1 by reacting it with 3-benzyloxycarbonylpropionaldehyde (intermediate 2) in a suitable organic medium, and then with tris(tert-butyl) phosphite to give intermediate 3;
c) Removal of phthalic groups to give corresponding diamine (intermediate 4) by reaction, for example, with hydrazine;
d) N-alkylation of the diamine 4 by reaction with a suitable halo acetate, such as, for example, tert-butyl bromoacetate, to give intermediate 5. This reaction is carried out in an organic solvent preferably selected from acetonitrile, ethyl acetate, and in the presence of a suitable tertiary amine such as, for example, diisopropylethylamine;
e) Debenzylation by catalytic hydrogenation of the intermediate 5 and isolation of the hexaester monocarboxylic acid 6. In a preferred process this hydrogenation is carried out in an organic solvent such as, for example, THF and catalysed by 10% Pd-C;
f) reaction of the hexaester monocarboxylic acid 6 with a suitable amino compound (compound 7) and isolation of the corresponding amide (derivative 8). In a preferred process said reaction is performed in presence of HATU (O-(7-Azabenzotriazol-1-yl)-N,N,Nxe2x80x2,Nxe2x80x2-tetramethyluronium hexafluoro-phosphate);
g) Deprotection of the acidic functions of the hexaester and recovery of the acid chelating agent (compound 10). In one preferred process of the invention the deprotection of the acidic functions is performed on the hexaester derivative prepared at step e) (intermediate 6) to give, for example, the acid chelating agent of EXAMPLE 7, disclosed later on in the experimental section of the invention. In the process of Scheme 1 bis, otherwise, the deprotection includes a first debenzylation step, by catalytic hydrogenation, of the benzylester contained in the amido derivative 8 and a second step, including the deprotection of the residual acidic functions of the hexaester 9, to give the chelating agent 10. The hydrogenation is preferentially carried out in an organic solvent such as, for example, THF and catalysed by 10% Pd-C. The subsequent deprotection can be performed, for example, by reacting the hexaester 9 with trifluoroacetic acid.
On the other hand, compounds of general formula (III) are preferably prepared according to the following Scheme 2
wherein R1 has the values defined above for compounds (I).
The synthetic process of Scheme 2 comprises the following steps:
a) Preparation of aminomethylphosphonic acid bis tert-butyl ester (bis N-alkyl) derivative (intermediate 3) by reacting bis tert-butyl phosphite, suitably activated (intermediate 1), with aminal (intermediate 2). In particular, in the process of the invention, the phosphonic acid tert-butyl ester is advantageously activated for example with Me3SiCl in a reaction carried out in organic solvent and in the presence of an amine, such as triethylamine. The resulting trimethylsilyl derivative is reacted with intermediate 2 obtained from 2-benzylaminoethanol and aqueous formaldehyde. This reaction is activated by the presence of a catalytic amount of a lanthanide triflate. Particularly preferred is ytterbium triflate. In the process of the invention, the resulting trimethylsilyl derivative is not isolated but it is directly transformed into the corresponding hydroxy derivative by treatment with a suitable aqueous acid, such as aqueous acetic acid.
b) Catalytic hydrogenation of intermediate 3. In the preferred process this reaction is carried out in alcoholic medium and catalyzed by Pd(OH)2/C.
c) N-alkylation of the resulting compound from step b), (intermediate 4), by reacting it with a suitable haloacetate, such as tert-butyl bromoacetate. This reaction is carried out in an organic solvent preferably selected from acetonitrile, ethyl acetate, and in the presence of a buffer solution pH 8.
d) Transformation of the isolated aminoalcohol (intermediate 5) into the corresponding bromo derivative by reacting it with methanesulfonyl chloride and a brominating agent such as lithium bromide. This reaction is carried out in an organic solvent selected from THF, acetonitrile, ethyl acetate, under nitrogen atmosphere and in the presence of an amine selected from triethylamine, diisopropylethylamine, at temperatures ranging from 20 to xe2x88x925xc2x0 C.
e) Condensation of the bromo derivative (intermediate 6) with a convenient amino acid suitably esterified (intermediate 7) and isolation of the polyester (intermediate 8). Said reaction is advantageously carried out in an organic solvent preferably selected from acetonitrile, THF, ethyl acetate and in the presence of a buffer solution pH 8.
f) Deprotection of the acidic functions of the polyester and isolation of the chelating agent (compound 9). The deprotection is, for example, obtained by reacting the polyester with an acid selected from HCl, H2SO4, in an aqueous mixture of an organic solvent such as dioxane.
The compounds of this invention have a wide range of applications, since they can be used for intravasal, (for instance i.v., intraarterial, intracoronaric, intraventricular administration and so on), intrathecal, intraperitoneal, intralymphatic and intracavital administrations. Furthermore, the compounds are suitable for the oral or enteral administration, and therefore, specifically for the imaging of the gastrointestinal tract.
For the parenteral administration they can preferably be formulated as sterile aqueous solutions or suspensions, whose pH can range from 6.0 to 8.5.
These aqueous solutions or suspensions can be administered in concentrations ranging between 0.002 and 1.0 M. These formulations can be lyophilized and supplied as they are to be reconstituted before use.
For the gastrointestinal use or for injection in body cavities, these agents can be formulated as a solution or suspension optionally containing suitable excipients in order, for example, to control viscosity.
For the oral administration, they can be formulated according to preparation methods routinely used in the pharmaceutical technique or as coated formulations to gain additional protection against the stomach acidic pH, thus preventing the chelated metal ion from release, which takes place particularly at the pH values typical of gastric juices.
Other excipients, such as sweeteners and/or flavouring agents, can also be added, according to known techniques of pharmaceutical formulations.
The solutions or suspensions of the compounds of this invention can also be formulated as aerosols to be used in aerosol-bronchography and instillation.
The compounds of the present invention can optionally be chemically conjugated to suitable macromolecules, targeting vectors or inglobated into suitable carriers.
For example they can also be encapsulated in liposomes or they can be constituents of their chemical structure and used as uni- or multilamellar vesicles.
A non-limiting list of preferred compounds of the invention is reported in the following, to better exemplify the wide applicative potential of the invention. 